In the field of point-of-care clinical chemistry, many test devices utilize direct protein-analyte affinity to express positive results for such analytes; for example protein analysis, drug screening, and medical diagnostics, among others (Camarero, 2008). In the case of lateral flow devices, a variety of proteins may be fixed on a membrane and when an analyte-containing sample flows over a specific protein for which the analyte has affinity, binding is effected. Additionally, a label is used, typically adhered to the analyte, to effect signal generation for interpretation of positive results.
Increased sensitivity can be achieved by mixing in solution the analyte and analyte-specific protein before subjecting the sample to lateral flow. Analyte to protein binding can be more complete when pre-mixing is used due to the additional binding time and better availability of protein binding sites in the absence of the constraints provided by a membrane. When analyte and protein are pre-mixed and already bound, a subsequent capture of protein-analyte on a membrane is required. Nucleic acid oligomer affinity pairs represent one method of capture wherein one half of the pair is bound to the protein and the corresponding complementary oligomer is bound to the membrane. Affinity ligand pairs have been used in various applications, including multiple sectors of life sciences across a range of pharmaceutical, biochemical, biophysical and diagnostic applications (Laitinen et al., 2006). Several types of affinity ligand pairs are being used to different extents: antibodies and their fragments; receptors and their ligands; avidin/biotin systems; textile and biomimetic dyes; (oligo)peptides; antisense peptides; chelated metal cations; lectins and phenylboronates; protein A and G; calmodulin; DNA; sequence-specific DNA; (oligo)nucleotides; heparin; and digoxigenin. (Labrou and Clonis, 1994; Hart and Basu, 2009). The importance of site-specific immobilization of proteins on solid supports in diagnostics has been reviewed (Carmerero, 2008).
While a single affinity pair of nucleic acid oligomers can be designed to function as a capture system on solid support or lateral flow, the design of larger numbers of affinity pairs for use in the same assay requires careful attention to avoid cross-interaction of non-specific pairs. In general, longer oligomers are more likely to contain cross-interacting subsequences which can adversely affect assay specificity. Short oligomers are therefore preferable to avoid such cross-interactions. Lateral flow immunoassays, nucleic acid lateral flow immunoassays (NALFIA), and nucleic acid lateral flow assays (NALF) have been reviewed (Postuma-Trumpie et al., 2009, this publication is hereby incorporated herein by reference in its entirety).
Because short natural DNA and RNA nucleic acid oligomers have relatively weak binding, alternate forms of nucleic acids with stronger binding characteristics are preferable. Examples include locked nucleic acid (LNA) and chimera LNA/DNA polymers, which demonstrate fast second-order kinetics with increased stability when hybridized to DNA targets (Christensen et al, 2001). The thermodynamic nearest neighbor parameters for LNA bases allow the Tm prediction of LNA:DNA and chimera-LNA-DNA:DNA duplexes (McTigue et al., 2004). Pyranosyl nucleic acid (pRNA), and 3-deoxypyranosyl nucleic acid (pDNA) are polymers that preferentially pair with complementary pRNA or pDNA versus natural RNA and DNA sequences (Schlonvogt et al. 1996; U.S. Pat. No. 7,153,955). Pentopyranosyl nucleic acid preparation and use for the production of a therapeutic, diagnostic and/or electronic component has been described (U.S. Pat. No. 6,506,896). These pRNA and pDNA nucleic acids also exhibit faster binding kinetics versus natural DNA which presents advantages when running an assay which requires binding in a mobile environment.
The design of multiple nucleic acid sequences with the same Tm poses special challenges for use in applications such as microarrays and nano-fabrications. It is essential to prevent undesired hybridizations. It is required that multiple nucleic acid sequences need to be designed that do not hybridize non-specifically with each other (Tanaka et al., 2005).
The Tm, of 6-mer oligomers were compared in Table 3 where it was shown that pRNA and pDNA respectively have Tms of 41.4° C. and 31.2° C. in a buffer containing NaCl and MgCl2. In comparison the Tm of a corresponding DNA oligomer is 5.5° C. (calculated using MGB Eclipse™ Design Software 2.0. Epoch Biosciences, Bothell, Wash.) demonstrating the dramatically increased stability of the pRNA and pDNA duplexes. It was experimentally observed that an increased non-specific hybridization between multiple designed oligomers occurred as duplex stability increased (larger Tms).
The problem of designing a system composed of nucleic acid pairs which exhibit orthogonality (the lack of cross reactivity with non-complementary pairs) is addressed in the present application and by the present invention. The inherent ability of nucleic acids to cross-pair among non-complementary strands, despite multiple mismatches, is effectively enhanced in nucleic acid systems such as pRNA and pDNA which exhibit stronger pairing per nucleotide versus natural DNA or RNA. Because empirical testing of 4n oligomers (where n represents oligo length) is cumbersome and impractical, rules were applied in a design algorithm which limits the output of orthogonal pairs. As noted in the literature, random design of DNA 25-mers resulted in 10 million oligomers (Xu et al., 2009) which were culled using stability and potential cross-reactivity. The present application describes how application of stability and potential cross-reactivity rules to the design process results in an algorithm which greatly improves efficiency in generating such orthogonal nucleic acid pairs.
Site specific immobilization of proteins to solid supports is of great importance in numerous applications including medical diagnostics, drug screening and protein analysis, among others (Camerio, 2007). Köhn (2009) reviewed approaches for site specific immobilization. Chen et al., (2011) reported that, despite the tremendous progress in developing bioorthogonal chemistry for site-specific labeling and surface immobilization of proteins over the past decade, the demand for new bioorthogonal methods with improved kinetics and selectivities remains high.
The orthogonal pDNA ligand pairs described in the present application are ideally suited to site-specifically immobilize proteins and peptides to solid surfaces in a similar fashion as illustrated in FIG. 12. Bioconjugation techniques are well known in the art (Hermanson, 1996). As illustrated in FIG. 12, one partner of an affinity pair can be covalently attached to a solid support with known chemistries or through a polymer in a site-specific fashion. The complementary partner of the affinity pair can be covalently attached to a protein or peptide. The solid phase immobilized affinity ligand efficiently captures the affinity ligand-modified protein or peptide. The capture protein or peptide can then be used in variety of biological assays as immunosensors (Shen Z et al., 2008), diagnostic immuno assays, protein:protein base screening (Tomizaki et al., 2010) and protein drug screening (Maynard et al., 2009).
Detection of ligand captured targets is achieved by using numerous available detection reagents such as fluorescent- and colored-dyes, fluorescent- and colored-beads, nanoparticles, enzymes and the like. Oligomer affinity pairs disclosed in this application are ideally suited to prepare detection reagents to detect capture targets which include nucleic acids, proteins, peptides and small molecules. A similar approach to the one described in Example 3 can be used or alternatively the ligand recognizing the target can be derivatized with a pDNA affinity pair member as taught in the art (Hermanson 1996, pages 639-67) while the complementary affinity pair member is covalently attached to a detection moiety which may include fluorophores, fluorescent beads, colored beads, nanoparticles, enzymes and the like, each containing a reactive group for covalent attachment. Affinity pairs are ideal in instances where more than one detection moiety is required. The detection of influenza A, influenza B and respiratory syncytial virus with nanoparticle is reported in respiratory samples (Jannetto et al., 2010).